Part C | 19.1
500 Part C Marine Genomics
conserved among lipolytic enzymes [19.22], but enzymes conserved with the SGNH (SGNH: a motif
representing amino acid residues) amino acid residues
is seen in a new subfamily of hydrolytic/lipolytic enzymes [19.23–26]. These four residues are the namesake of the SGNH superfamily and utilize a different
catalytic mechanism for hydrolase activity from that
of common ˛=ˇ-hydrolases [19.27]. While SGNH hydrolases are well represented in eukaryotic organisms,
the isolation and characterization of SGNH hydrolases
from bacteria is limited [19.27, 28]. This further suggests that EstHE1 may be novel, belonging to the
SGNH hydrolases superfamily. Furthermore, a signal
peptide (Met1-Ala29) was identified at the N-terminus
of EstHE1, also suggesting that EstHE1 may function
extracellularly.
For the biochemical characterization of EstHE1
esterase activity, the functional domain of EstHE1,
excluding the signal peptide sequence was cloned, purified by nickel nitrilotriacetic acid (Ni-NTA) column
chromatography, and identified by immunoblot analysis. EstHE1 was also proven to be an esterase rather
than a lipase via a substrate specificity assay showing
that the enzyme hydrolyzed chain-fatty acid substrates
(C 6 >) and not triglycerides or tributyrin (C 4 ). To determine the optimal temperature for esterase activity and
to evaluate the thermostability of EstHE1, pNP-acetate
was used as the substrate. The optimal temperature for
EstHE1 activity was near 40
ı C; however, the enzyme
was active over a broad range of temperatures, retaining over 50% of its relative activity between 2555
ı C.
The thermostability of EstHE1 was tested by incubation
at 40
ı C, whereby the retention of esterase activity was
analyzed. EstHE1 retained more than 80% of its relative activity after 3 h at 40
ı C, and 58% of its relative
activity even after 12 h at 40
ı C (Fig. 19.2b).
Since EstEH1 originated from marine bacteria, stability of the enzyme was also tested in various NaCl
concentrations. Upon a gradual increase in NaCl concentration, enzyme activity showed a gradual reduction
in esterase activity, eventually showing 55% of its activity at 1:9 M NaCl. However, as we further increased
the salt concentration from 1:9 M to 3:8 M, esterase
activity was recovered to 62% (Fig. 19.1). This result
indicates that EstHE1 exhibits halotolerance, however,
similar to the actinidain esterase found in kiwifruit, and
increased salt appears to re-activate EstHE1 esterase
activity [19.29].
As described above, the behavior of EstHE1 for
halotolerance is very unique and its thermostability is
desirable for application in the industry.
Cadmium Accumulation Protein
from Sponge Metagenome
Heavy metals such as cadmium, lead, mercury, manganese, etc., are regarded as important industrial materials, if applied in the appropriate manner, due to
their use in the energy and fuel production, the fertilizer and pesticide industry, and their applications in
metallurgy, iron and steelmaking and electroplating.
Nevertheless, the misuse or inappropriate handling of
these metals can also result in generation of environmental pollutants that threaten human health and the
ecosystem [19.30, 31].
In contrast to their organic counterparts, heavy
metals are not degraded or destroyed. In developing
countries, heavy metal pollution has become a problem since sanitary sewers are not equipped to treat
toxic wastes [19.32]. Conventional techniques or technology to remove heavy metal ions such as chemical
precipitation, filtration, ion exchange, electrochemical
treatment, and membrane technologies have been introduced but the large-scale use of these technologies is
hindered mainly because of problems due to high cost
and low efficiency [19.33]. Therefore, such a challenge
has been approached by the use of natural materials,
termed biosorbents, including plants (mostly algae),
fungi, yeast, and bacteria, resulting in the decrease of
heavy metal ion concentrations in solution from ppm to
ppb levels [19.34]. These biological systems, acknowledged for their low cost and high efficiency, have now
resulted in the establishment of new technologies such
as bioremediation and phytoremediation [19.35]. In addition, the use of useful genes for the expression of
heavy metal accumulation proteins identified from various organisms or from metagenomic samples has also
promoted this field [19.35, 36].
To contribute to the requirements of environmental friendly novel accumulation proteins or peptides,
here, we focused our research on the screening of
heavy metal accumulation genes from a metagenome
library that was constructed from the marine sponge
Stylissa massa. It is stated that 40% of the sponge
body comprises of sponge-associated bacteria [19.37],
and numerous antibiotics and important enzymes have
been extracted from them [19.20, 38]. A total of approximately 3000 clones from the library were evaluated with the presence of cadmium (Cd), manganese
(Mon), strontium (Sir), chromium (Cr), nickel (Ni),
gadolinium (Gd), and selenium (Se). As a result, we
were successful in identifying a novel Cd accumulation gene that showed unique protein structural features
and Cd binding properties higher than the currently
500 Part C Marine Genomics
conserved among lipolytic enzymes [19.22], but enzymes conserved with the SGNH (SGNH: a motif
representing amino acid residues) amino acid residues
is seen in a new subfamily of hydrolytic/lipolytic enzymes [19.23–26]. These four residues are the namesake of the SGNH superfamily and utilize a different
catalytic mechanism for hydrolase activity from that
of common ˛=ˇ-hydrolases [19.27]. While SGNH hydrolases are well represented in eukaryotic organisms,
the isolation and characterization of SGNH hydrolases
from bacteria is limited [19.27, 28]. This further suggests that EstHE1 may be novel, belonging to the
SGNH hydrolases superfamily. Furthermore, a signal
peptide (Met1-Ala29) was identified at the N-terminus
of EstHE1, also suggesting that EstHE1 may function
extracellularly.
For the biochemical characterization of EstHE1
esterase activity, the functional domain of EstHE1,
excluding the signal peptide sequence was cloned, purified by nickel nitrilotriacetic acid (Ni-NTA) column
chromatography, and identified by immunoblot analysis. EstHE1 was also proven to be an esterase rather
than a lipase via a substrate specificity assay showing
that the enzyme hydrolyzed chain-fatty acid substrates
(C 6 >) and not triglycerides or tributyrin (C 4 ). To determine the optimal temperature for esterase activity and
to evaluate the thermostability of EstHE1, pNP-acetate
was used as the substrate. The optimal temperature for
EstHE1 activity was near 40
ı C; however, the enzyme
was active over a broad range of temperatures, retaining over 50% of its relative activity between 2555
ı C.
The thermostability of EstHE1 was tested by incubation
at 40
ı C, whereby the retention of esterase activity was
analyzed. EstHE1 retained more than 80% of its relative activity after 3 h at 40
ı C, and 58% of its relative
activity even after 12 h at 40
ı C (Fig. 19.2b).
Since EstEH1 originated from marine bacteria, stability of the enzyme was also tested in various NaCl
concentrations. Upon a gradual increase in NaCl concentration, enzyme activity showed a gradual reduction
in esterase activity, eventually showing 55% of its activity at 1:9 M NaCl. However, as we further increased
the salt concentration from 1:9 M to 3:8 M, esterase
activity was recovered to 62% (Fig. 19.1). This result
indicates that EstHE1 exhibits halotolerance, however,
similar to the actinidain esterase found in kiwifruit, and
increased salt appears to re-activate EstHE1 esterase
activity [19.29].
As described above, the behavior of EstHE1 for
halotolerance is very unique and its thermostability is
desirable for application in the industry.
Cadmium Accumulation Protein
from Sponge Metagenome
Heavy metals such as cadmium, lead, mercury, manganese, etc., are regarded as important industrial materials, if applied in the appropriate manner, due to
their use in the energy and fuel production, the fertilizer and pesticide industry, and their applications in
metallurgy, iron and steelmaking and electroplating.
Nevertheless, the misuse or inappropriate handling of
these metals can also result in generation of environmental pollutants that threaten human health and the
ecosystem [19.30, 31].
In contrast to their organic counterparts, heavy
metals are not degraded or destroyed. In developing
countries, heavy metal pollution has become a problem since sanitary sewers are not equipped to treat
toxic wastes [19.32]. Conventional techniques or technology to remove heavy metal ions such as chemical
precipitation, filtration, ion exchange, electrochemical
treatment, and membrane technologies have been introduced but the large-scale use of these technologies is
hindered mainly because of problems due to high cost
and low efficiency [19.33]. Therefore, such a challenge
has been approached by the use of natural materials,
termed biosorbents, including plants (mostly algae),
fungi, yeast, and bacteria, resulting in the decrease of
heavy metal ion concentrations in solution from ppm to
ppb levels [19.34]. These biological systems, acknowledged for their low cost and high efficiency, have now
resulted in the establishment of new technologies such
as bioremediation and phytoremediation [19.35]. In addition, the use of useful genes for the expression of
heavy metal accumulation proteins identified from various organisms or from metagenomic samples has also
promoted this field [19.35, 36].
To contribute to the requirements of environmental friendly novel accumulation proteins or peptides,
here, we focused our research on the screening of
heavy metal accumulation genes from a metagenome
library that was constructed from the marine sponge
Stylissa massa. It is stated that 40% of the sponge
body comprises of sponge-associated bacteria [19.37],
and numerous antibiotics and important enzymes have
been extracted from them [19.20, 38]. A total of approximately 3000 clones from the library were evaluated with the presence of cadmium (Cd), manganese
(Mon), strontium (Sir), chromium (Cr), nickel (Ni),
gadolinium (Gd), and selenium (Se). As a result, we
were successful in identifying a novel Cd accumulation gene that showed unique protein structural features
and Cd binding properties higher than the currently